Protective effects of the combination Bifidobacterium longum plus lactoferrin against NSAID-induced enteropathy.

OBJECTIVES: Nonsteroidal anti-inflammatory drugs can exert detrimental effects in the lower digestive tract. The aim of this study was to examine the protective effects of a combination of the probiotic Bifidobacterium longum BB536 (Bifidobacterium) with the prebiotic lactoferrin in a rat model of diclofenac-induced enteropathy. METHODS: Enteropathy was induced in 40-wk-old male rats by intragastric diclofenac (4 mg/kg twice daily for 14 d). Lactoferrin (100 mg/kg twice daily), Bifidobacterium (2.5 × 106 CFU/rat twice daily) or their combination were administered 1 h before diclofenac. At the end of treatments, the ileum was processed for the evaluation of histologic damage, myeloperoxidase (MPO) and malondialdehyde (MDA) levels, as well as the expression of Toll-like receptors 2 and 4 (TLR-2/-4) and the activation of downstream signaling molecules (MyD88 and nuclear factor [NF]-κB p65). Blood hemoglobin and fecal calprotectin were also assessed. RESULTS: Diclofenac induced intestinal damage, along with increments of MPO and MDA, overexpression of TLR-2, TLR-4, MyD88, and NF-κB p65, increased fecal calprotectin and decreased blood hemoglobin levels. Lactoferrin or Bifidobacterium alone prevented diclofenac-induced enteric damage, and the changes in blood hemoglobin, MPO, MDA, fecal calprotectin, and NF-κB p65. Bifidobacterium, but not lactoferrin, decreased TLR-4 expression, although none of them affected MyD88 overexpression. TLR-2 expression was slightly enhanced by all treatments. The combined administration of lactoferrin and Bifidobacterium reduced further the intestinal damage, and restored MPO and blood hemoglobin levels. CONCLUSIONS: Diclofenac induced ileal mucosal lesions by activation of inflammatory and pro-oxidant mechanisms. These detrimental actions were prevented by the combination of lactoferrin with Bifidobacterium likely through the modulation of TLR-2/-4/NF-κB proinflammatory pathways.

Pathophysiology of NSAID-Associated Intestinal Lesions in the Rat: Luminal Bacteria and Mucosal Inflammation as Targets for Prevention.

Non-steroidal anti-inflammatory drugs (NSAIDs) can damage the small intestine, mainly through an involvement of enteric bacteria. This study examined the pathophysiology of NSAID-associated intestinal lesions in a rat model of diclofenac-enteropathy and evaluated the effect of rifaximin on small bowel damage. Enteropathy was induced in 40-week old male rats by intragastric diclofenac (4 mg/kg BID, 14 days). Rifaximin (delayed release formulation) was administered (50 mg/kg BID) 1 h before the NSAID. At the end of treatments, parameters dealing with ileal damage, inflammation, barrier integrity, microbiota composition, and TLR-NF-κB-inflammasome pathway were evaluated. In addition, the modulating effect of rifaximin on NLRP3 inflammasome was tested in an in vitro cell system. Diclofenac induced intestinal damage and inflammation, triggering an increase in tissue concentrations of tumor necrosis factor and interleukin-1ß, higher expression of TLR-2 and TLR-4, MyD88, NF-κB and activation of caspase-1. In addition, the NSAID decreased ileal occludin expression and provoked a shift of bacterial phyla toward an increase in Proteobacteria and Bacteroidetes abundance. All these changes were counterbalanced by rifaximin co-administration. This drug was also capable of increasing the proportion of Lactobacilli, a genus depleted by the NSAID. In LPS-primed THP-1 cells stimulated by nigericin (a model to study the NLRP3 inflammasome), rifaximin reduced IL-1ß production in a concentration-dependent fashion, this effect being associated with inhibition of the up-stream caspase-1 activation. In conclusion, diclofenac induced ileal mucosal lesions, driving inflammatory pathways and microbiota changes. In conclusion, rifaximin prevents diclofenac-induced enteropathy through both anti-bacterial and anti-inflammatory activities.

Aging Modulates the Influence of Arginase on Endothelial Dysfunction in Obesity.

Objective- Arginase can reduce NO availability. In this study, we explored arginase as a determinant of endothelial dysfunction in small arteries from obese patients and its relationship with aging and microvascular remodeling. Approach and Results- Small arteries were dissected after subcutaneous fat biopsies and evaluated on a pressurized micromyograph. Endothelium-dependent vasodilation was assessed by acetylcholine, repeated under L-NAME ( N G-nitro-L-arginine-methyl ester), N(ω)-hydroxy-nor-l-arginine (arginase inhibitor) and gp91ds-tat (NADPH [nicotinamide adenine dinucleotide phosphate oxidase] oxidase inhibitor) in vessels from young (age <30 years) control and obese and old (>30 years) control and obese subjects. Media-lumen ratio and amount of vascular wall fibrosis were used as markers of vascular remodeling. Amount of vascular superoxide anions and NO production were determined with immunofluorescence, whereas arginase expression was quantified using Western blot and quantitative polymerase chain reaction. Obese and older age groups had lower vascular NO, as well as higher media-lumen ratio, wall fibrosis, intravascular superoxide, and blunted inhibitory effect of L-NAME on acetylcholine versus controls and younger age groups. N(ω)-hydroxy-nor-l-arginine restored the acetylcholine-induced vasodilation in young and, to a lesser extent, in old obese subjects. This effect was abolished by addition of L-NAME. Gp91ds-tat increased the vasodilatory response to N(ω)-hydroxy-nor-l-arginine in old obese. Superoxide anions and arginase I/II levels were higher in the vascular wall of obese versus controls. Conclusions- Arginase contributes to microvascular endothelial dysfunction in obesity. Its impact is reduced by aging because of higher levels of vascular oxidative stress. Obesity is accompanied by accelerated microvascular remodeling, the extent of which is related to the amount of arginase in the vascular wall.

The flavonoid compound apigenin prevents colonic inflammation and motor dysfunctions associated with high fat diet-induced obesity.

BACKGROUND AND PURPOSE: Apigenin can exert beneficial actions in the prevention of obesity. However, its putative action on obesity-associated bowel motor dysfunctions is unknown. This study examined the effects of apigenin on colonic inflammatory and motor abnormalities in a mouse model of diet-induced obesity. EXPERIMENTAL APPROACH: Male C57BL/6J mice were fed with standard diet (SD) or high-fat diet (HFD). SD or HFD mice were treated with apigenin (10 mg/Kg/day). After 8 weeks, body and epididymal fat weight, as well as cholesterol, triglycerides and glucose levels were evaluated. Malondialdehyde (MDA), IL-1ß and IL-6 levels, and let-7f expression were also examined. Colonic infiltration by eosinophils, as well as substance P (SP) and inducible nitric oxide synthase (iNOS) expressions were evaluated. Motor responses elicited under blockade of NOS and tachykininergic contractions were recorded in vitro from colonic longitudinal muscle preparations. KEY RESULTS: When compared to SD mice, HFD animals displayed increased body weight, epididymal fat weight and metabolic indexes. HFD mice showed increments in colonic MDA, IL-1ß and IL-6 levels, as well as a decrease in let-7f expression in both colonic and epididymal tissues. HFD mice displayed an increase in colonic eosinophil infiltration. Immunohistochemistry revealed an increase in SP and iNOS expression in myenteric ganglia of HFD mice. In preparations from HFD mice, electrically evoked contractions upon NOS blockade or mediated by tachykininergic stimulation were enhanced. In HFD mice, Apigenin counteracted the increase in body and epididymal fat weight, as well as the alterations of metabolic indexes. Apigenin reduced also MDA, IL-1ß and IL-6 colonic levels as well as eosinophil infiltration, SP and iNOS expression, along with a normalization of electrically evoked tachykininergic and nitrergic contractions. In addition, apigenin normalized let-7f expression in epididymal fat tissues, but not in colonic specimens. CONCLUSIONS AND IMPLICATIONS: Apigenin prevents systemic metabolic alterations, counteracts enteric inflammation and normalizes colonic dysmotility associated with obesity.

Colonic motor dysfunctions in a mouse model of high-fat diet-induced obesity: an involvement of A2B adenosine receptors.

Adenosine A2B receptors (A2BR) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A2BR in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A2BR localization was examined by immunofluorescence. The role of A2BR in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A2BR were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A2BR antagonist MRS1754 enhanced electrically induced NK1-mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A2BR ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A2BR expression. A2BR, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.

Alteration of colonic excitatory tachykininergic motility and enteric inflammation following dopaminergic nigrostriatal neurodegeneration.

BACKGROUND: Parkinson's disease (PD) is frequently associated with gastrointestinal (GI) symptoms, including constipation and defecatory dysfunctions. The mechanisms underlying such disorders are still largely unknown, although the occurrence of a bowel inflammatory condition has been hypothesized. This study examined the impact of central dopaminergic degeneration, induced by intranigral injection of 6-hydroxydopamine (6-OHDA), on distal colonic excitatory tachykininergic motility in rats. METHODS: Animals were euthanized 4 and 8 weeks after 6-OHDA injection. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. SP, tachykininergic NK1 receptor, and glial fibrillary acidic protein (GFAP) expression, as well as the density of eosinophils and mast cells in the colonic wall, were examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay), TNF, and IL-1ß (ELISA assay) levels were also examined. The polarization of peritoneal macrophages was evaluated by real-time PCR. RESULTS: In colonic preparations, electrically and SP-evoked tachykininergic contractions were increased in 6-OHDA rats. Immunohistochemistry displayed an increase in SP and GFAP levels in the myenteric plexus, as well as NK1 receptor expression in the colonic muscle layer of 6-OHDA rats. MDA, TNF, and IL-1ß levels were increased also in colonic tissues from 6-OHDA rats. In 6-OHDA rats, the number of eosinophils and mast cells was increased as compared with control animals, and peritoneal macrophages polarized towards a pro-inflammatory phenotype. CONCLUSIONS: The results indicate that the induction of central nigrostriatal dopaminergic degeneration is followed by bowel inflammation associated with increased oxidative stress, increase in pro-inflammatory cytokine levels, activation of enteric glia and inflammatory cells, and enhancement of colonic excitatory tachykininergic motility.

Fibrotic and Vascular Remodelling of Colonic Wall in Patients with Active Ulcerative Colitis.

BACKGROUND AND AIMS: Intestinal fibrosis is a complication of inflammatory bowel disease [IBD]. Although fibrostenosis is a rare event in ulcerative colitis [UC], there is evidence that a fibrotic rearrangement of the colon occurs in the later stages. This is a retrospective study aimed at examining the histopathological features of the colonic wall in both short-lasting [SL] and long-lasting [LL] UC. METHODS: Surgical samples of left colon from non-stenotic SL [≤ 3 years, n = 9] and LL [≥ 10 years, n = 10] UC patients with active disease were compared with control colonic tissues from cancer patients without UC [n = 12] to assess: collagen and elastic fibres by histochemistry; vascular networks [CD31/CD105/nestin] by immunofluorescence; parameters of fibrosis [types I and III collagen, fibronectin, RhoA, alpha-smooth muscle actin [α-SMA], desmin, vimentin], and proliferation [proliferating nuclear antigen [PCNA]] by western blot and/or immunolabelling. RESULTS: Colonic tissue from both SL-UC and LL-UC showed tunica muscularis thickening and transmural activated neovessels [displaying both proliferating CD105-positive endothelial cells and activated nestin-positive pericytes], as compared with controls. In LL-UC, the increased collagen deposition was associated with an up-regulation of tissue fibrotic markers [collagen I and III, fibronectin, vimentin, RhoA], an enhancement of proliferation [PCNA] and, along with a loss of elastic fibres, a rearrangement of the tunica muscularis towards a fibrotic phenotype. CONCLUSIONS: A significant transmural fibrotic thickening occurs in colonic tissue from LL-UC, together with a cellular fibrotic switch in the tunica muscularis. A full-thickness angiogenesis is also evident in both SL- and LL-UC with active disease, as compared with controls.

Small bowel protection against NSAID-injury in rats: Effect of rifaximin, a poorly absorbed, GI targeted, antibiotic.

Nonsteroidal anti-inflammatory drugs, besides exerting detrimental effects on the upper digestive tract, can also damage the small and large intestine. Although the underlying mechanisms remain unclear, there is evidence that enteric bacteria play a pivotal role. The present study examined the enteroprotective effects of a delayed-release formulation of rifaximin-EIR (R-EIR, 50mg/kg BID, i.g.), a poorly absorbed antibiotic with a broad spectrum of antibacterial activity, in a rat model of enteropathy induced by indomethacin (IND, 1.5mg/kg BID for 14 days) administration. R-EIR was administered starting 7 days before or in concomitance with IND administration. At the end of treatments, blood samples were collected to evaluate hemoglobin (Hb) concentration (as an index of digestive bleeding). Small intestine was processed for: (1) histological assessment of intestinal damage (percentage length of lesions over the total length examined); (2) assay of tissue myeloperoxidase (MPO) and TNF levels, as markers of inflammation; (3) assay of tissue malondialdehyde (MDA) and protein carbonyl concentrations, as an index of lipid and protein peroxidation, respectively; (4) evaluation of the major bacterial phyla. IND significantly decreased Hb levels, this effect being significantly blunted by R-EIR. IND also induced the occurrence of lesions in the jejunum and ileum. In both intestinal regions, R-EIR significantly reduced the percentage of lesions, as compared with rats receiving IND alone. Either the markers of inflammation and tissue peroxidation were significantly increased in jejunum and ileum from IND-treated rats. However, in rats treated with R-EIR, these parameters were not significantly different from those observed in controls. R-EIR was also able to counterbalance the increase in Proteobacteria and Firmicutes abundance induced by INDO. To summarize, R-EIR treatment significantly prevents IND-induced intestinal damage, this enteroprotective effect being associated with a decrease in tissue inflammation, oxidative stress and digestive bleeding as well as reversal of NSAID-induced alterations in bacterial population.

The AMPK enzyme-complex: from the regulation of cellular energy homeostasis to a possible new molecular target in the management of chronic inflammatory disorders.

INTRODUCTION: Adenosine monophosphate-activated protein kinase (AMPK), known as an enzymatic complex that regulates the energetic metabolism, is emerging as a pivotal enzyme and enzymatic pathway involved in the regulation of immune homeostatic networks. It is also involved in the molecular mechanisms underlying the pathophysiology of chronic inflammatory diseases. AREAS COVERED: AMPK is expressed in several immune cell types including macrophages, lymphocytes, neutrophils and dendritic cells, and governs a broad array of cell functions, which include cytokine production, chemotaxis, cytotoxicity, apoptosis and proliferation. Based on its wide variety of immunoregulatory actions, the AMPK system has been targeted to reveal its impact on the course of immune-related diseases, such as atherosclerosis, psoriasis, joint inflammation and inflammatory bowel diseases. EXPERT OPINION: The identification of AMPK subunits responsible for specific anti-inflammatory actions and the understanding of the underlying molecular mechanisms will promote the generation of novel AMPK activators, endowed with improved pharmacodynamic and pharmacokinetic profiles. These new tools will aid us to utilize AMPK pathway activation in the management of acute and chronic inflammatory diseases, while minimizing potential adverse reactions related to the effects of AMPK on metabolic energy.

Ghrelin restores nitric oxide availability in resistance circulation of essential hypertensive patients: role of NAD(P)H oxidase.

AIMS: We assessed whether acute intra-arterial infusion of exogenous ghrelin can improve endothelial dysfunction by restoring nitric oxide (NO) availability in the forearm microcirculation of essential hypertensive patients. The effect of ghrelin on endothelial dysfunction (pressurized myograph), vascular oxidative stress generation (fluorescent dihydroethidium), and phosphorylation of p47phox (western blot), an index of NAD(P)H oxidase activation, in isolated small arteries taken from essential hypertensive patients (subcutaneous biopsy) were also investigated. METHODS AND RESULTS: In 18 normotensive control subjects and 18 essential hypertensive patients, we studied the forearm blood flow (strain-gauge plethysmography) response to intra-arterial acetylcholine, repeated under NO synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) or the antioxidant ascorbic acid. The protocol was repeated at the end of exogenous ghrelin intra-arterial infusion. In hypertensive patients, ghrelin normalized the blunted response to acetylcholine, restored the inhibiting effect of l-NMMA and abrogated the potentiating effect of ascorbic acid on acetylcholine. In controls, ghrelin failed to modify these vascular responses. In hypertensive patients, ghrelin decreased venous levels of malondialdehyde, lipoperoxide, and interleukin-6, and concomitantly increased endogenous antioxidant capacity. Small vessels from hypertensive patients showed an enhanced intravascular oxidative stress, which was strongly and similarly decreased by incubation with ghrelin, the NAD(P)H oxidase inhibitor gp91 ds-tat, or both. Ghrelin also normalized the overexpression of p47 phosphorylation and restored the NO availability in small vessels from hypertensive patients. CONCLUSIONS: Exogenous ghrelin increases endothelial dysfunction by restoring NO availability in the forearm microcirculation of essential hypertensive patients, an effect ascribable to an antioxidant effect via inhibition of NAD(P)H oxidase activation.